The long-term goals of this research project are to delineate the biochemical events involved in glucose-induced insulin secretion by the Beta-cell. A subcellular approach developed in our laboratory directed at obtaining and characterizing a plasma membrane enriched fraction from islets has allowed us to focus on the central role the plasma membrane plays in mediating cation fluxes and interacting with insulin secretagogues. In this project we will determine the role of calmodulin in modulating a high affinity Ca2++Mg2+-ATPase activity localized in the plasma membrane and determine the direct effect of insulin secretagogues on this activity. The reaction mechanism of the Ca2++Mg2+-ATPase will be studied by characterizing both Ca2+-dependent phosphoprotein intermediates and performing binding assays of 125I-calmodiun for identification of this enzyme activity. We have demonstrated ATP-dependent Ca2+-transport in this plasma membrane preparation and will correlate this transport process with the Ca2++Mg2+-ATPase activity, as well as evaluate this preparation for Na+/K+ exchange. This subcellular approach will be used to describe further a calmodulin activated protein kinase activity identified in islet cell particulate membranes which phosphorylates an endogenous protein substrate M.W. 57,000. Since the protein kinase activity is only activated in the combined presence of Ca2+ and calmodulin, it may function in stimulus-secretion coupling. In this project, the recognition of D-glucose by the Beta-cell will also be approached by evaluating specific chemical analogues of this major insulin secretagogue based on 3-dimensional structure analysis for in vitro insulin release activity.